Endoplasmic reticulum-associated degradation (ERAD) is a process in which ER proteins are degraded, either because they are misfolded or because their degradation is physiologically regulated. The hallmark substrate of this process is HMG-CoA reductase, an enzyme catalyzing the rate-limiting step in cholesterol synthesis. In addition to degrading metabolically-regulated or misfolded proteins, ERAD functions as stress-response system to alleviate ER stress. While its normal function is to target misfolded or regulated proteins, ERAD is also exploited by bacterial and viral pathogens to gain access to the cytosol. During normal ERAD, protein substrates are selected within the ER lumen, moved across the membrane to the cytosol (retrotranslocation), where they are polyubiquitinated, extracted from the membrane and degraded by the proteasome. How physiologically- regulated substrates are selected for degradation and the specific cellular pathways under the control of ERAD are ambiguous. Recent work has demonstrated that a central, conserved ubiquitin ligase called Hrd1, forms a ubiquitin-gated protein-conducting channel that that is sufficient to allow retrotranslocation of misfolded lumenal and integral membrane proteins. The autoubiquitination-gating mechanism presents a conundrum; under normal circumstances, ubiquitination will result in degradation of the modified protein. However, Hrd1 is relatively stable meaning there are unidentified mechanisms in place to protect autoubiquitinated Hrd1 from degradation and to reverse the ubiquitin-gated activation. Along with the regulation of Hrd1, many associated processes including substrate selection by ERAD are mysterious. The goals of this proposal are to 1) define how the ERAD system is regulated, 2) determine how the ERAD system recognizes its targets, 3) define the features of targets directing them to ERAD (the degrons), 4) understand how the membrane contributes to ERAD function, and 5) identify the cellular function of other (non-ERAD) integral membrane protein quality control systems. We will use a multifaceted approach with biochemistry, cell biology, genetics, and proteomics to address these central questions in membrane-associated protein quality control. We will leverage our unique in vivo and in vitro assays (and continue to design innovative assays) to dissect the basic mechanisms of these eukaryotic systems. A mechanistic understanding of how the systems function (including ERAD) and the processes they regulate will establish these systems as viable therapeutic targets in medically relevant pathways of protein misfolding, protein misregulation, and pathogen hijacking.